Recombinant vector containing immunogenic protein of african swine fever virus, recombinant bacteria and use thereof

ABSTRACT

A recombinant vector containing the immunogenic protein of African swine fever virus, a recombinant bacterium and use thereof, and relates to the technical field of gene recombination. The recombinant vector can be used to construct a recombinant  Lactobacillus  expressing the immunogenic protein of African swine fever virus, and after mixing the  Lactobacillus  solution that can secrete protein p72 and protein p54, respectively, an oral live bacterial preparation for preventing African swine fever can be prepared. The oral live bacteria preparation prepared by the disclosure can safely, effectively and quickly prevent the infection of African swine fever virus to pigs, and does not contain an immune process.

CROSS REFERENCE TO RELATED APPLICATION

This patent application claims the benefit and priority of ChinesePatent Application No. 202010339345.5 filed on Apr. 26, 2020, thedisclosure of which is incorporated by reference herein in its entiretyas part of the present application.

REFERENCE TO SEQUENCE LISTING

A Sequence Listing in ASCII text format, submitted pursuant to 37 C.F.R.§ 1.821, entitled SEQUENCE-LISTING.txt, 6 kilobytes in size, created onMay 14, 2021 and filed via EFS-Web, is hereby incorporated by referencein its entirety.

TECHNICAL FIELD

The disclosure belongs to the technical field of gene recombination, andparticularly relates to a recombinant vector containing immunogenicprotein of African swine fever virus, a recombinant bacteria and usethereof.

BACKGROUND ART

African swine fever (ASF) is an acute, febrile and highly contagiousanimal infectious disease. The morbidity and mortality of ASF can reachas high as 100%, which is the number one killer in swine industry. Atpresent, there is no commercial vaccine available. African swine fevervirus (ASFV) is the only member of African swine fever virus familyAfrican swine fever virus genus, which is 175-215 nm in diameter,icosahedron symmetrical, and with capsule membrane coated on thenucleocapsid. The genome of ASFV is a double strand linear DNA with asize of 170-190kb.

African swine fever virus can cause highly contact transmission betweendomestic pigs and various wild boars, mainly entering pigs through themouth and upper respiratory system, causing infection in nasopharynx ortonsil, then spreading rapidly to mandibular lymph nodes and invadingthe whole body through lymph and blood.

Since there is no commercial vaccine against ASFV on the market, thesafest, most economical and effective prevention and control method isbiosafety prevention and control method, the principle of which is toblock the contact between virus and organism. However, none of theexisting methods can guarantee that the virus will no longer come intocontact with the body.

SUMMARY

In view of this, the purpose of the present disclosure is to provide arecombinant vector containing the immunogenic protein of African swinefever virus, a recombinant bacteria and use thereof, to construct aLactobacillus expression system expressing p72 and p54 proteins ofAfrican swine fever virus, and to provide a theoretical basis for thedevelopment of a mucosal infection blocking oral agent for blockingvirus infection.

In order to achieve the above purpose of the disclosure, the presentdisclosure provides the following technical solution:

The present disclosure provides a recombinant vector containingimmunogenic protein of African swine fever virus, wherein therecombinant vector takes Lactobacillus expression vector pVE5523 asbasic vector, and the nucleotide sequence encoding the immunogenicprotein of African swine fever virus is cloned between EcoRV and SalIrestriction sites of the basic vector.

In some embodiments, the immunogenic protein of African swine fevervirus comprises p72 protein and p54 protein of African swine fever virusof Jilin strain in China. The nucleotide sequence encoding the p72protein of the African swine fever virus of Jilin strain in China is setforth in SEQ ID NO:1, and the nucleotide sequence encoding the p54protein of the African swine fever virus of Jilin strain in China is setforth in SEQ ID NO:2.

The present disclosure provides a recombinant Lactobacillus expressingthe immunogenic protein of African swine fever virus, wherein therecombinant Lactobacillus comprises the above recombinant vector.

The present disclosure also provides a constructing method of therecombinant Lactobacillus, wherein comprising:

-   -   (1) respectively cloning the nucleotide sequence encoding the        p72 protein of African swine fever virus and the nucleotide        sequence encoding the p54 protein of African swine fever virus        into the pVE5523 Lactobacillus expression vector to construct        recombinant plasmids pVE5523-ASFV-p72 and pVE5523-ASFV-p54;    -   (2) respectively transforming the recombinant plasmids        pVE5523-ASFV-p72 and pVE5523-ASFV-p54 into Lactobacillus        competent cells to obtain recombinant Lactobacillus expressing        p72 protein of African swine fever virus and recombinant        Lactobacillus expressing p54 protein of African swine fever        virus.

The present disclosure also provides an oral live bacteria preparationfor preventing African swine fever infection, wherein the activeingredients of the oral live bacteria preparation comprise recombinantLactobacillus expressing p72 protein of African swine fever virus andrecombinant Lactobacillus expressing p54 protein of African swine fevervirus constructed by the above constructing method.

In some embodiments, the live bacteria ratio of the recombinantLactobacillus expressing p72 protein of African swine fever virus to therecombinant Lactobacillus expressing p54 protein of African swine fevervirus is (0.8−1.2)×108 cfu: (0.8−1.2)×108 cfu.

The disclosure provides a recombinant vector containing immunogenicprotein of African swine fever virus, which can be used to constructrecombinant Lactobacillus expressing immunogenic protein of Africanswine fever virus, and after mixing the recombinant Lactobacillusexpressing African swine fever virus antigen proteins p72 and p54, anoral live bacteria preparation for preventing African swine fever virusinfection is prepared. In the present disclosure, after pigs take thepreparation, the antigen protein (the mixture of protein p72 and p54)secreted by Lactobacillus in the preparation adheres to the mucousmembrane on the surface of organism cells, and forms an antigen proteinbiofilm on the mucous membrane surface. The antigen protein can becombined with the virus binding site on the target cells to seal thevirus receptor protein site on the mucous membrane surface, thus playingthe role of ecological occupation. When the live viruses in theenvironment invade the body, the virus binding site on the target cellhas been completely blocked by antigen protein biofilm, and the viruscan not bind to the virus binding site on the target cell, thuseffectively blocking the combination of the virus with the receptor onthe cell surface and preventing African swine fever.

Compared with vaccines, the oral preparation prepared by the disclosureis safer, more effective and faster. The safety of the oral preparationdisclosed by the disclosure is manifested in that the effectivecomponents only secrete viral functional proteins, no viral genes exist,and no virus variation can be caused. The effectiveness is manifested inthat the effective components of the oral preparation only secreteprotective antigens, act on the mucosal site on the body surface, andcover the mucosal surface where the target cells of African swine fevervirus are located. When the virus invades, the binding site betweenantigen protein and mucosal surface cells is blocked and sealed, thusblocking the virus infection path. The rapidity is manifested in thatthe secreted protein expressed by Lactobacillus directly preempts thereceptor that the virus binds to the target cell, thus blocking thevirus infection, and no immune response process is needed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plasmid structure diagram of the recombinant vectorpVE5523-ASFV-p72 in the present disclosure;

FIG. 2 is a plasmid structure diagram of the recombinant vectorpVE5523-ASFV-p54 in the present disclosure;

FIG. 3 is an amplification curve for verifying p72 expression in thepresent disclosure;

FIG. 4 is an amplification curve for verifying p54 expression in thepresent disclosure.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The disclosure will be further explained with embodiments below.

The present disclosure provides a recombinant vector containingimmunogenic protein of African swine fever virus, wherein therecombinant vector takes Lactobacillus expression vector pVE5523 asbasic vector, and the nucleotide sequence encoding the immunogenicprotein of African swine fever virus is cloned between EcoRV and SalIrestriction sites of the basic vector.

The immunogenic protein of African swine fever virus comprises p72protein and p54 protein of African swine fever virus of Jilin strain inChina. The nucleotide sequence encoding the p72 protein of the Africanswine fever virus of Jilin strain in China is set forth in SEQ ID NO:1,and the nucleotide sequence encoding the p54 protein of the Africanswine fever virus of Jilin strain in China is set forth in SEQ ID NO:2.The nucleotide sequence set forth in SEQ ID NO:1 is cloned between theEcoRV and SalI restriction sites of the basic vector to form arecombinant vector pVE5523-ASFV-p72, and the structure of therecombinant plasmid is shown in FIG. 1. The nucleotide sequence setforth in SEQ ID NO:2 is cloned between the EcoRV and SalI restrictionsites of the basic vector to form a recombinant vector pVE5523-ASFV-p54,and the structure of the recombinant plasmid is shown in FIG. 2. Thepresent invention has no special limitation on the constructing methodof the recombinant vector, and the conventional constructing method ofthe recombinant vector will do.

The present disclosure provides a recombinant Lactobacillus expressingthe immunogenic protein of African swine fever virus, wherein therecombinant Lactobacillus comprises the above recombinant vector.

The present disclosure also provides a constructing method of therecombinant Lactobacillus, wherein comprising:

-   -   (1) respectively cloning the nucleotide sequence encoding the        p72 protein of African swine fever virus and the nucleotide        sequence encoding the p54 protein of African swine fever virus        into the pVE5523 Lactobacillus expression vector to construct        recombinant plasmids pVE5523-ASFV-p72 and pVE5523-ASFV-p54;    -   (2) respectively transforming the recombinant plasmids        pVE5523-ASFV-p72 and pVE5523-ASFV-p54 into Lactobacillus        competent cells to obtain recombinant Lactobacillus expressing        p72 protein of African swine fever virus and recombinant        Lactobacillus expressing p54 protein of African swine fever        virus.

Before performing the cloning step, the p72 and p54 gene sequences ofAfrican swine fever virus of Jilin strain in China searched in GenBankare preferably optimized to form sequences set forth in SEQ ID NO:1 andSEQ ID NO:2, and then the cloning is performed.

When the cloning is performed, the vector pVE5523 is preferably digestedwith SalI/EcoRV, and the sequences set forth in SEQ ID NO:1 and SEQ IDNO:2 are digested with the same enzyme. After ligating the abovedigested fragments, the recombinant plasmids pVE5523-ASFV-p72 andpVE5523-ASFV-p54 shown in FIG. 1 and FIG. 2 are formed.

In the disclosure, the recombinant plasmids are used for transformingthe ATCC393 Lactobacillus casei competent cells by electrotransformationto obtain the recombinant Lactobacillus. After obtaining the recombinantLactobacillus, the recombinant Lactobacillus are preferably amplified inMRS liquid medium, and the recombinant plasmid is extracted forfluorescence quantitative PCR detection. The primers and amplifiedsequences used for fluorescence quantitative PCR detection in thisdisclosure are as follows:

Detection of p72 fluorescence quantitative PCR:

P72 forward primer (SEQ ID NO: 3): AGTTCGGATGTCACAACGCTTG;P72 reverse primer (SEQ ID NO: 4): TTTGCTTTGGTGCGGCTTGT;P72 amplified sequence (SEQ ID NO: 5):AGTTCGGATGTCACAACGCTTGTGCGCAAATTTT GCATCCCAGGGGATAAAATGACTGGATATAAGCACTTGGTTGGCCAGGAGGTATCGGTGGAGGGAACC AGTGGCCCTCTCCTATGCAACATTCATGATTTGCACAAGCCGCACCAAAGCAAA;

P54 fluorescence quantitative PCR:

P54 forward primer (SEQ ID NO: 6): AGCCACTCCACAACCAGGTAC;P54 reverse primer (SEQ ID NO: 7): GCCCTCCAGTTGCCATGATTAG;P54 amplified sequence (SEQ ID NO: 8):AGCCACTCCACAACCAGGTACCTCTAAACCGGCTGGAGCCACTACAGGCAACGTAGGCAAGCCAATTACAGACAGGCCAGTTGCCATGAATAGGCCAGTTACGAACAGCTCGGTCGCGGACAGGCCAGTTATGAACAACCCAGT TACGGACAGACTAATCATGGCAACTGGAGGGC.

The recombinant Lactobacillus prepared by the constructing method cansecrete recombinant immunogenic proteins p72 and p54 of African swinefever virus respectively according to different recombinant plasmids.

The present disclosure also provides an oral live bacteria preparationfor preventing African swine fever infection, wherein the activeingredients of the oral live bacteria preparation comprise recombinantLactobacillus expressing p72 protein of African swine fever virus andrecombinant Lactobacillus expressing p54 protein of African swine fevervirus constructed by the above constructing method.

In the oral live bacteria preparation, the live bacteria ratio of therecombinant Lactobacillus expressing p72 protein of African swine fevervirus to the recombinant Lactobacillus expressing p54 protein of Africanswine fever virus is (0.8-1.2)x10⁸ cfu: (0.8-1.2)×10⁸ cfu, morepreferably 1×10⁸ cfu.

In the following, the recombinant vector containing the immunogenicprotein of African swine fever virus, recombinant bacteria and usethereof provided by the present disclosure will be described in detailwith reference to the Examples, but they should not be understood aslimiting the scope of protection of the present disclosure.

EXAMPLE 1 Acquisition of p72 and p54 Gene Sequences of African SwineFever Virus

p54 protein of African swine fever virus exists in the inner capsule ofvirus particles, which is one of the main structural proteins and strongimmunogenic proteins of ASFV, and participates in the adsorption andentry of virus to target cells. The p72 and p54 gene sequences (P72:GenBank: MK189456.1; P54: GenBank: MK214679.1) of African swine fevervirus of Jilin strain in China in GenBank were optimized and modified,then synthesized by Nanjing Genescript Biotechnology Co., Ltd, and thebase sequences were set forth in SEQ ID NO:1 and SEQ ID NO:2.

EXAMPLE 2

Acquisition of Recombinant Expression Vectors pVE5523-ASFV-p72 andpVE5523-ASFV-p54

1 Materials and Methods 1.1 Materials and Sources

The restriction enzymes SalI and EcoRV were all purchased from NEBcompany, and Taq enzyme, dNTP, DNA Marker DL2000, DL15000, Agarose GelDNA Purification Kit and Mini BEST Plasmid purification kit werepurchased from Dalian Takara company,and the cloning vector pVE5523 wasprovided by Nanjing Genescript Biotechnology Co., Ltd.

1.2 Testing Method

The fragments of cloning vector pVE5523 digested by SalI/EcoRV wereligated with fragments of p72 and p54 gene digested by the sameSalI/EcoRV enzyme, after the electrotransformation, the recombinantplasmid was extracted and sent to Nanjing Genescript Biotechnology Co.,Ltd for sequencing verification.

2 Testing Results

Sequencing results of recombinant plasmid: the recombinant plasmid aftergene sequencing was compared with the inserted p72 and p54 genefragments, and the sequencing results were consistent with expectations,indicating that the synthesized p72 and p54 gene fragments weresuccessfully inserted into Lactobacillus vector pVE5523, and therecombinant plasmid was successfully constructed, the positive plasmidswere named pVE5523-ASFV-p72 and pVE5523-ASFV-p54 respectively.

EXAMPLE 3 Preparation and Detection of p72 and p54 Genes ExpressingAfrican Swine Fever Virus 1 Materials and Methods 1.1 Materials andSources

Erythromycin (Emr) was purchased from Biodee Biotechnology Co., Ltd.

1.2 Testing Method

Electrotransformation of target genes in Lactobacillus ATCC393 andscreening of resistant strains: the electrotransformed LactobacillusATCC393 was spread on MRS solid culture plate containing 5 μg/mlerythromycin, the plate was cultured in incubator at 30° C. for 72hours, and the colonies on the plate were selected and inoculated intoMRS liquid culture medium containing 5 μg/ml erythromycin, and culturedat 30° C. for 72 hours. The plasmids in bacteria were extracted andidentified by fluorescence quantitative PCR. The identified primers andamplified sequences are as follows:

Detection of p72 fluorescence quantitative PCR:

P72 forward primer (SEQ ID NO: 3): AGTTCGGATGTCACAACGCTTG;P72 reverse primer (SEQ ID NO: 4): TTTGCTTTGGTGCGGCTTGT;P72 amplified sequence (SEQ ID NO: 5):AGTTCGGATGTCACAACGCTTGTGCGCAAATTTTG CATCCCAGGGGATAAAATGACTGGATATAAGCACTTGGTTGGCCAGGAGGTATCGGTGGAGGGAACCAGT GGCCCTCTCCTATGCAACATTCATGATTTGCACAAGCCGCACCAAAGCAAA;

P54 fluorescence quantitative PCR:

P54 forward primer (SEQ ID NO: 6): AGCCACTCCACAACCAGGTAC;P54 reverse primer (SEQ ID NO: 7): GCCCTCCAGTTGCCATGATTAG;P54 amplified sequence (SEQ ID NO: 8):AGCCACTCCACAACCAGGTACCTCTAAACCGGCTGGAGCCACTACAGGCAACGTAGGCAAGCCAATTACAGACAGGCCAGTTGCCATGAATAGGCCAGTTACGAACAGCTCGGTCGCGGACAGGCCAGTTATGAACAACCCAGT TACGGACAGACTAATCATGGCAACTGGAGGGC.

2 Testing Results

The amplified recombinant plasmids were detected by fluorescencequantitative PCR, and the amplification curves are shown in FIG. 3 andFIG. 4 respectively. The positive recombinant plasmids have typicalamplification curve with CT values ranging from 19 to 22, while ATCC393competent control has no amplification curve, indicating that therecombinant plasmids pVE5523-ASFV-p54 and pVE5523-ASFV-p72 have beensuccessfully transformed into ATCC393 competent cells.

EXAMPLE 4 Method for Culturing Recombinant Lactobacillus ExpressionSystem

The recombinant Lactobacillus expression system was inoculated intoLactobacillus MRS liquid culture medium with 1% inoculation amount, andthe fermentation broth was obtained at 35° C. for 72 hours.

Viable Bacteria Count of Recombinant Lactobacillus

With flat surface dispersion method:

1. Numbering: 9 sets of sterile MRS solid agar culture plates weremarked as 10⁻⁴, 10⁻⁵ and 10⁻⁶ (3 sets for each dilution) with a markerrespectively. Another 6 test tubes containing 4.5mL sterile water weretaken and marked as 10⁻¹, 10⁻², 10⁻³, 10⁻⁴, 10⁻⁵ and 10⁻⁶ in turn.

2. Diluting: 0.5 ml of Lactobacillus suspension (sample to be tested)that well mixed was suck to the 10⁻¹ test tube with a 1 mL pipette,which was a 10 times diluent. The 10⁻¹ test tube was placed on the testtube oscillator for oscillation, to make the bacterial liquid mixedevenly. Another lml pipette was used to insert into the 10⁻¹ test tubeto blow and suck the bacterial suspension back and forth for threetimes, to further disperse and mix the bacteria. 0.5 ml bacterial liquidin the 10⁻¹ test tube was suck to the the 10⁻² test tube with thepipette used in the previous step, obtaining a 100 times diluent, andthe rest can be deduced by analogy.

3. Sampling and coating: 0.2 ml of diluted bacterial suspensions weretaken from 10⁻⁴, 10⁻⁵ and 10⁻⁶ test tubes respectively, and put into thecorresponding numbered sterile agar medium plate, the bacterial solutionwas uniformly dispersed in the agar medium with a sterile glass coatingrod, and the bacteria was cultivated in a constant temperature incubatorat 37° C.

4. Accounting: After culturing for 48 hours, the agar medium plate wastaken out for counting the colonies, and the average number of colonieson three plates with the same dilution was calculated, the calculatingformula was as follows:

Colony forming unit per milliliter (cfu)=the average number of coloniesrepeated three times with the same dilutionxdilution times×5.

EXAMPLE 5

The Lactobacillus that can secrete recombinant p72 and p54 proteinsprepared in Example 4 was diluted into 0.8-1.2×10⁸ cfu/ml bacterialliquid and mixed in the ratio of 1:1. The mixed bacterial solution wasadministered to SPF New Zealand rabbits and SD rats in the form of oralliquid. Meanwhile, the negative control group A was given the samevolume of normal saline and the control group B was given the samevolume of MRS culture solution. Four New Zealand rabbits and SD rats ineach group were observed for 2 weeks, and no abnormal phenomena such asabnormal body temperature and allergy occurred in 24 animals. Therefore,the Lactobacillus that can secrete recombinant p72 and p54 proteinprepared by the disclosure is safe and has no side effect.

EXAMPLE 6

There were about 300 pigs in a farm in Taihu County, Anhui Province,with sizes ranging from 20 kg to 100 kg, which were divided into threebreeding areas, some pigs in the second breeding areas had become illand died one after another. On Jan. 23, 2020, the test of recombinantLactobacillus preparation was carried out in this farm, the test lastedfor 21 days and was divided into three groups, the pigs were givenorally at a dose of 5 ml/head. The control group was set in the firstbreeding area with 112 pigs in total, the test group was set in thesecond and third breeding area. Before the experiment, the pigs in thefirst and third breeding area were in a healthy state, and the secondbreeding area was in an outbreak state of African swine fever before thetest was carried out on January 23.

The testing results are shown in Table 1, wherein the number of pigs inthe control group ranged from 112 to 66, and 46 pigs died, with amortality rate of 41.07%. Because the second breeding area was in thedisease stage before the experiment, the number decreased from 45 to 28after using the recombinant Lactobacillus preparation, and 17 died, witha mortality rate of 37.78%. One pig died during the test in the thirdbreeding area, with a mortality rate of 1.08%. In the whole test stage,18 pigs died in the test group (the second and third breeding areas),with a mortality rate of 13.04%. The results showed that the recombinantLactobacillus preparation had good effect.

TABLE 1 Comparison of testing results of recombinant LactobacillusControl group Test group Time/Group Breeding area 1 Breeding area 2Breeding area 3 January 23- The number The second The number February 13of pigs in the breeding of pigs control group area was ranged from 93ranged from in the disease to 92, and 112 to 66, and stage before onepig died 46 pigs died the experiment, the number decreased from 45 to 28after using the recombinant Lactobacillus preparation, and 17 diedMortality rate 41.07% 13.04%

EXAMPLE 7

An African swine fever epidemic occurred in a farm in Yiyang City, HunanProvince in 2019, more than 400 fattening pigs were treated harmlesslyand the farm was completely disinfected. In April 2020, a total of 120nursing pigs and fattening pigs were introduced, and 402 pigs wereintroduced on May 27, the pathogen detection of African swine fever wasnot conducted before the introduction. Among the pigs introduced inApril, some pigs suffered from non-eating, poor mental state and thelike, and 1 pig died. Then 40 pigs were randomly sampled to detectpathogen of African swine fever, and 2 nursing pigs were suspected ofAfrican swine fever, and farmers immediately isolated the suspected pigsand strengthen the biosafety measures of the farm, two suspected pigsdied within a week, and then died one after another. On May 21st, 500 mlof the recombinant Lactobacillus preparation prepared in Example 4 wasdissolved in 30 kg of water, and mixed with 25 kg of feed (feedingamount of 100 pigs per day), and fed twice a day, 2-3 times a week.After the product being used, the overall health status of pigs improvedobviously, and no death occurred.

TABLE 2 Collection table of pig farm use effect of recombinantLactobacillus products Introduction Before using After the productinformation the product is used 5# On Apr. 1, Since April The healthstatus Building 2020, 60 13, deaths of pigs improved pigs were occurredone obviously, and no introduced after another, death happened with 9deaths. 9# On Apr. 1, Since April The health status Building 2020, 6019, deaths of pigs improved pigs were occurred one obviously, and nointroduced after another, death happened with 18 deaths.

All the 402 pigs introduced in May grew well using the scheme of Example4, and no pigs were infected.

EXAMPLE 8

A pig farm in Jinhua City, Zhejiang Province, was non-pestilencepositive in November 2019. At present, there were 380 sows and about3,900 fattening pigs in stock. All sows and fattening pigs wereadministrated with the recombinant Lactobacillus products prepared inExample 4. From the beginning of the year to May, 10 ml per pig wastaken orally, once every 3 days. From May 17, 500 ml of the recombinantLactobacillus preparation prepared in Example 4 was dissolved in 30 kgof water, and mixed with 25 kg of feed (feeding amount of 100 pigs perday), and fed twice a day, 2-3 times a week. After the Spring Festivalin 2020, there was an outbreak of non-plague in Wucheng District,Jinhua, and by August 10th, everything in the pig farm was normal.

The above are only the preferred embodiments of the present disclosure.It should be pointed out that for those of ordinary skill in the art,without departing from the principle of the present disclosure, severalimprovements and modifications can be made, and these improvements andmodifications should also be regarded as the protection scope of thepresent disclosure.

SEQUENCE LISTING

-   <110> Changsha Lvye Biotechnology Co., Ltd.-   <120> RECOMBINANT VECTOR COMPRISING IMMUNOGENIC PROTEIN OF AFRICAN    -   SWINE FEVER VIRUS, RECOMBINANT BACTERIA AND USE THEREOF-   <130> GWPCTP202103493-   <160> 8-   <170> PatentIn version 3.5-   <210> 1-   <211> 2128-   <212> DNA-   <213> Artificial Sequence-   <220>-   <223> The nucleotide sequence encoding the p72 protein of the    African swine fever virus of Jilin strain in China-   <400> 1

gtcgacatgg gtgtactgct cacacagagg acgctgctca gtctggtcct tgcactcctg   60tttccaagca tggcgagcat gcatatgaaa gggatgcac gtggcccaacc tgcagtagtg  120ctggccaaca gccggggtgt tgccagcttt gtgtgtgagt atgggtctgc aggcaaagct  180gccgaggtcc gggtgacagt gctgcggcgg gccggcagcc agatgactga agtctgtgcc  240gcgacatata ctgtggagga tgagttgacc ttccttgatg actctacatg cactggcacc  300tccaccgaaa acaaagtgaa cctcaccatc caagggctga gagccgtgga cactgggctc  360tacatctgca aggtggagct cctgtaccca ccaccctact atgtgggtat gggcaacggg  420acccagattt atgtcattga tccagaacca tgcccagatt ctgatggtgg cggtggctcg  480ggcggtggtg gatctggtgg cggcggatct acaaagacca aaccaccatc ccccatatcg  540ccaggctccg aagtggccgg gtcctcggtc ttcatcttcc ctccaaaacc caaggacacc  600ctcatgatct cccagacccc cgaggtcacg tgcgtggtgg tggacgtcag caaggagcac  660gccgaggtcc agttctcctg gtacgtggac ggcgtagagg tgcacacggc cgagacgaga  720ccaaaggagg agcagttcaa cagcacctac cgtgtggtca gcgtcctgcc catccagcac  780caggactggc tgaaggggaa ggagttcaag tgcaaggtca acaacgtaga cctcccagcc  840cccatcacga ggaccatctc caaggctata gggcagagcc gggagccgca ggtgtacacc  900ctgcccccac ccgccgagga gctgtccagg agcaaagtca ccgtaacctg cctggtcatt  960ggcttctacc cacctgacat ccatgttgag tggaagagca acggacagcc ggagccagag 1020ggcaattacc gcaccacccc gccccagcag gacgtggacg ggaccttctt cctgtacagc 1080aagctcgcgg tggacaaggc aagatgggat catggagaaa catttgagtg tgcggtgatg 1140cacgaggctc tgcacaacca ctacacccag aagtccatct ccaagactca gggtaaacct 1200cctccatacc agcctctcgg cggcggcggc agcgaattcg gatcccatat ggacaagatt 1260atattggccc aagacttgct gaatagcagg atctctaaca ttaaaaatgt gaacaaaagt 1320tatgggaaac ccgatcccga acccactttg agtcaaatcg aagaaacaca tttggtgcat 1380tttaatgcgc attttaagcc ttatgttcca gtagggtttg aatacaataa agtacgcccg 1440catacgggta cccccacctt gggaaacaag cttacctttg gtattcccca gtacggagac 1500tttttccatg atatggtggg ccatcatata ttgggtgcat gtcattcatc ctggcaggat 1560gctccgattc agggcacgtc ccagatgggg gcccatgggc agcttcaaac gtttcctcgc 1620aacggatatg actgggacaa ccaaacaccc ttagagggcg ccgtttacac gcttgtagat 1680ccttttggaa gacccattgt acccggcaca aagaatgcgt accgaaactt ggtttactac 1740tgcgaatacc ccggagaacg actttatgaa aacgtaagat tcgatgtaaa tggaaattcc 1800ctagacgaat atagttcgga tgtcacaacg cttgtgcgca aattttgcat cccaggggat 1860aaaatgactg gatataagca cttggttggc caggaggtat cggtggaggg aaccagtggc 1920cctctcctat gcaacattca tgatttgcac aagccgcacc aaagcaaacc tattcttacc 1980gatgaaaatg atacgcagcg aacgtgtagc cataccaacc cgaaatttct ttcacagcat 2040tttcccgaga actctcacaa tatccaaaca gcaggtaaac aagatattac tcctatcacg 2100gacgcaacgt atctggacat aagatatc                                    2128

-   <210> 2-   <211> 574-   <212> DNA-   <213> Artificial Sequence-   <220>-   <223> The nucleotide sequence encoding the p54 protein of the    African swine fever virus of Jilin strain in China-   <400> 2

gtcgacatgg attctgaatt ttttcaaccc gtttatccgc ggcattatgg cgaatgtttg  60tcaccaacct ctacaccgag cttcttctcc acacatatgt gtactattct cgttgctatc 120gtggtcttaa tcattattat catcgttcta atttatctgt tttcttcaag aaagaaaaaa 180gctgctgccc ccgctattga ggaggaagat atacagttta taaatcctta tcaagatcag 240cagtgggcag gagccactcc acaaccaggt acctctaaac cggctggagc cactacaggc 300aacgtaggca agccaattac agacaggcca gttgccatga ataggccagt tacgaacagc 360tcggtcgcgg acaggccagt tatgaacaac ccagttacgg acagactaat catggcaact 420ggagggccag cggccgcaag tgctccttcg gatgagcttt atacaacagc cactactcag 480aacactgctt cacaaacaat gccggctgtt gaagctctac ggcaaagaag cacctataca 540cacaaagacc tggaaaactc cttgtaagat atca                             574

-   <210> 3-   <211> 22-   <212> DNA-   <213> Artificial Sequence-   <220>-   <223> p72 forward primer-   <400> 3

agttcggatg tcacaacgct tg 22

-   <210> 4-   <211> 20-   <212> DNA-   <213> Artificial Sequence-   <220>-   <223> p72 reverse primer-   <400> 4

tttgctttgg tgcggcttgt 20

-   <210> 5-   <211> 156-   <212> DNA-   <213> Artificial Sequence-   <220>-   <223> P72 amplified sequence-   <400> 5

agttcggatg tcacaacgct tgtgcgcaaa ttttgcatcc caggggataa aatgactgga  60tataagcact tggttggcca ggaggtatcg gtggagggaa ccagtggccc tctcctatgc 120aacattcatg atttgcacaa gccgcaccaa agcaaa                           156

-   <210> 6-   <211> 21-   <212> DNA-   <213> Artificial Sequence-   <220>-   <223> P54 forward primer-   <400> 6

agccactcca caaccaggta c 21

-   <210> 7-   <211> 22-   <212> DNA-   <213> Artificial Sequence-   <220>-   <223> P54 reverse primer-   <400> 7

gccctccagt tgccatgatt ag 22

-   <210> 8-   <211> 176-   <212> DNA-   <213> Artificial Sequence-   <220>-   <223> P54 amplified sequence-   <400> 8

agccactcca caaccaggta cctctaaacc ggctggagcc actacaggca acgtaggcaa  60gccaattaca gacaggccag ttgccatgaa taggccagtt acgaacagct cggtcgcgga 120caggccagtt atgaacaacc cagttacgga cagactaatc atggcaactg gagggc     176

1. A recombinant vector containing immunogenic protein of African swinefever virus, wherein the recombinant vector takes Lactobacillusexpression vector pVE5523 as basic vector, and the nucleotide sequenceencoding the immunogenic protein of African swine fever virus is clonedbetween EcoRV and SalI restriction sites of the basic vector.
 2. Therecombinant vector according to claim 1, wherein the immunogenic proteinof African swine fever virus comprises p72 protein and p54 protein ofAfrican swine fever virus of Jilin strain in China. The nucleotidesequence encoding the p72 protein of the African swine fever virus ofJilin strain in China is set forth in SEQ ID NO:1, and the nucleotidesequence encoding the p54 protein of the African swine fever virus ofJilin strain in China is set forth in SEQ ID NO:2.
 3. A recombinantLactobacillus expressing the immunogenic protein of African swine fevervirus, wherein the recombinant Lactobacillus comprises the recombinantvector of claim
 1. 4. A constructing method of recombinant Lactobacillusaccording to claim 3, wherein comprising: (1) respectively cloning thenucleotide sequence encoding the p72 protein of African swine fevervirus and the nucleotide sequence encoding the p54 protein of Africanswine fever virus into the pVE5523 Lactobacillus expression vector toconstruct recombinant plasmids pVE5523-ASFV-p72 and pVE5523-ASFV-p54;(2) respectively transforming the recombinant plasmids pVE5523-ASFV-p72and pVE5523-ASFV-p54 into Lactobacillus competent cells to obtainrecombinant Lactobacillus expressing p72 protein of African swine fevervirus and recombinant Lactobacillus expressing p54 protein of Africanswine fever virus.
 5. The constructing method according to claim 4,wherein before performing the cloning in step (1), the method furthercomprises optimizing the p72 and p54 gene sequences of African swinefever virus of Jilin strain in China searched in GenBank to formsequences set forth in SEQ ID NO:1 and SEQ ID NO:2, and then performingthe cloning.
 6. The constructing method according to claim 4, whereinafter obtaining the recombinant Lactobacillus in step (2), the methodfurther comprises propagating recombinant Lactobacillus in MRS liquidmedium, and extracting the recombinant plasmid for fluorescencequantitative PCR detection.
 7. The constructing method according toclaim 6, wherein the primer pairs used for fluorescent quantitative PCRdetection respectively comprise: primers for detecting p72 fluorescentquantitative PCR include p72 forward primers with the nucleotidesequence set forth in SEQ ID NO:3, and p72 reverse primers with thenucleotide sequence set forth in SEQ ID NO:4; primers for detecting p54fluorescent quantitative PCR include p54 forward primers with thenucleotide sequence set forth in SEQ ID NO:6, and p54 reverse primerswith the nucleotide sequence set forth in SEQ ID NO:7.
 8. A method forpreventing and/or treating African swine fever infection, whereincomprising administrating the recombinant vector of claim
 1. 9. A methodfor preventing and/or treating African swine fever infection, comprisingadministrating a drug to a subject in need thereof, wherein the drugcomprises the recombinant vector of claim
 1. 10. The method according toclaim 9, wherein the drug comprises oral live bacteria preparation. 11.An oral live bacteria preparation for preventing African swine feverinfection, wherein the active ingredients of the oral live bacteriapreparation comprise recombinant Lactobacillus expressing p72 protein ofAfrican swine fever virus and recombinant Lactobacillus expressing p54protein of African swine fever virus constructed by the constructingmethod of claim
 4. 12. The oral live bacteria preparation according toclaim 11, wherein the live bacteria ratio of the recombinantLactobacillus expressing p72 protein of African swine fever virus to therecombinant Lactobacillus expressing p54 protein of African swine fevervirus is (0.8−1.2)×10⁸ cfu: (0.8−1.2)×10⁸ cfu.
 13. The oral livebacteria preparation according to claim 12, wherein the live bacteriaratio of the recombinant Lactobacillus expressing p72 protein of Africanswine fever virus to the recombinant Lactobacillus expressing p54protein of African swine fever virus is 1×10⁸ cfu: 1×10⁸ cfu.
 14. Amethod for treating African swine fever, wherein comprising: allowinganimals to ingest the oral live bacteria preparation according to claim11, wherein the ingested amount is 5 ml per animal.
 15. The recombinantLactobacillus expressing the immunogenic protein of African swine fevervirus according to claim 3, wherein the recombinant Lactobacilluscomprises the recombinant vector of claim
 2. 16. A method for preventingand/or treating African swine fever infection, wherein comprisingadministrating the recombinant Lactobacillus of claim
 3. 17. A methodfor preventing and/or treating African swine fever infection, whereincomprising administrating the recombinant Lactobacillus obtained by theconstructing method of claim
 4. 18. A method for preventing and/ortreating African swine fever infection, comprising administrating a drugto a subject in need thereof, wherein the drug comprises the recombinantLactobacillus of claim
 3. 19. A method for preventing and/or treatingAfrican swine fever infection, comprising administrating a drug to asubject in need thereof, wherein the drug comprises the recombinantLactobacillus obtained by the constructing method of claim 4.